Over the past two decades, there has been an accumulation of evidence demonstrating the critical role of skeletal interstitial fluid flow in the viability, maintenance, and response to loading and unloading of bone. Our objective is to elucidate the mechanisms by which interstitial fluid flow (IFF) stimulates bone cells, using molecular, cell, and in vivo knockout and transgenic models. IFF is characterized by both steady and dynamic components driven by vascular pressure and mechanical loading. Even though bone cells respond to both flow components, we have demonstrated that the mechanotransduction pathways and the subsequent cellular and in vivo responses to these two mechanical stimuli differ. The overarching hypothesis is that dynamic or pulsatile flows result in an osteoblast mitogenic response while steady flow induces both an anti-resorptive skeletal response as well as osteoblast differentiation. To investigate this hypothesis, we propose the following the specific aims: 1) Test the hypothesis that oscillatory flow in vitro induces an osteoblast growth (mitogenic) response, while ramped steady flow induces osteoblast differentiation. Mitogenic indices (BrdU incorporation) and transcriptional activators (egr-1 and c-fos) and differentiation indices (bone sialoprotein, Cbfa-1, and p57Kip2) will be measured in osteoblasts and osteocytes. 2) Determine the mechanisms for the nitric oxide-related mechanochemical signal transduction pathways of pulsatile flow and ramped steady flow in osteoblasts and osteocytes. We will characterize which nitric oxide synthase (NOS) isoforms mediate the flow responses, and determine the mechanism of regulation of NOS activity. 3) Using our novel implantable microfluidic oscillatory pressure device, we will determine if oscillatory flow induces an anabolic response, while increased steady flow is anti-resorptive in the hindlimb suspended rat. And 4) using osteoblast-specific conditional knockout mice, determine the roles of NOS and caveolin in mediating the skeletal cellular responses to dynamic interstitial fluid flow. The proposed research will elucidate the basic mechanisms by which interstitial fluid flow acts on bone, and provide the basis for treatments based on the modulation of interstitial fluid flow to counter osteopenia of disuse. PUBLIC HEALTH RELEVANCE. There is mounting evidence that interstitial fluid flow (IFF) mediates the skeletal response to loading and unloading. The overarching hypothesis is that dynamic IFF results in an osteoblast mitogenic response while steady flow induces osteoblast differentiation. Using both in vitro and in vivo models, the proposed research will elucidate the basic mechanisms by which IFF acts on bone, and provide the basis for treatments based on the modulation of IFF to counter osteopenia of disuse.